ABSTRACT
GW501516, also known by the name of cardarine, is a synthetic peroxisome-proliferator-activated receptor delta (PPR-δ) agonist agent developed for applications in the treatment of metabolic disorders and cardiovascular diseases. A broad polymorph screening in various solvents and mixtures was completed in order to explore its capabilities to grow polymorphs. The crystal structures of four polymorphs were elucidated using single-crystal X-ray diffraction, while one structure was solved via a powder X-ray diffraction method. The solid state features (nature of intermolecular interactions) were investigated by computational methods. The polymorphs were further investigated by thermal DSC analysis and X-ray diffraction on powders. From a pharmaceutical perspective, the stability and solubility of the polymorphs were analyzed as well.
ABSTRACT
Bladder cancer aggressiveness is correlated with abnormal N-cadherin transmembrane glycoprotein expression. This protein is cleaved by the metalloprotease ADAM10 and the γ-secretase complex releasing a pro-angiogenic N-terminal fragment (NTF) and a proliferation-activating soluble C-terminal fragment (CTF2). Tetraspanin 15 (Tspan15) is identified as an ADAM10-interacting protein to induce selective N-cadherin cleavage. We first demonstrated, in invasive T24 bladder cancer cells, that N-cadherin was cleaved by ADAM10 generating NTF in the extracellular environment and leaving a membrane-anchored CTF1 fragment and that Tspan15 is required for ADAM10 to induce the selective N-cadherin cleavage. Targeting N-cadherin function in cancer is relevant to preventing tumor progression and metastases. For antitumor molecules to inhibit N-cadherin function, they should be complete and not cleaved. We first showed that the GW501516, an agonist of the nuclear receptor PPARß/δ, decreased Tspan15 and prevented N-cadherin cleavage thus decreasing NTF. Interestingly, the drug did not modify ADAM10 expression, which was important because it could limit side effects since ADAM10 cleaves numerous substrates. By targeting Tspan15 to block ADAM10 activity on N-cadherin, GW501516 could prevent NTF pro-tumoral effects and be a promising molecule to treat bladder cancer. More interestingly, it could optimize the effects of the N-cadherin antagonists those such as ADH-1 that target the N-cadherin ectodomain.
Subject(s)
ADAM10 Protein , Amyloid Precursor Protein Secretases , Antigens, CD , Cadherins , Dipeptides , Hydroxamic Acids , Membrane Proteins , Tetraspanins , Urinary Bladder Neoplasms , Humans , ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Cadherins/metabolism , Cell Line, Tumor , Membrane Proteins/metabolism , Neoplasm Invasiveness , Proteolysis/drug effects , Tetraspanins/metabolism , Tetraspanins/genetics , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/geneticsABSTRACT
OBJECTIVE To investigate the effects of the peroxisome proliferator-activated receptors δ (PPARδ) agonist GW501516 on the injury of pulmonary artery endothelial cells (PAECs) induced by hypoxia and its mechanism. METHODS The cytotoxic effects of GW501516 were observed by detecting the relative survival rate of PAECs; the protein expression of PPARδ was determined by Western blot assay. The cellular model of PAECs injury was established under hypoxic conditions; using antioxidant N-acetylcysteine (NAC) as positive control, the effects of GW501516 on cell injury and reactive oxygen species (ROS) production were investigated by detecting cell apoptotic rate, cell viability, lactate dehydrogenase (LDH) activity and ROS levels. Using nuclear factor erythroid 2-related factor 2(Nrf2) activator dimethyl fumarate (DMF) as positive control, PAECs were incubated with GW501516 and/or Nrf2 inhibitor ML385 under hypoxic conditions; the mechanism of GW501516 on PAECs injury induced by hypoxia was investigated by detecting cell injury (cell apoptosis, cell viability, LDH activity), the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), malondialdehyde (MDA) and ROS, the expressions of Nrf2, heme oxygenase-1 (HO-1) and cleaved-caspase-3 (C-caspase-3) protein. RESULTS The results demonstrated that hypoxia inhibited the protein expression of PPARδ (P<0.05), while GW501516 promoted the protein expression of PPARδ in hypoxia- exposed PAECs without obvious cytotoxic effects. GW501516 inhibited the apoptosis of PAECs, improved cell viability, and reduced LDH activity and ROS levels. GW501516 could up-regulate the protein expression of HO-1 in PAECs and the levels of SOD, GPx and CAT, while down-regulated the levels of MDA and ROS by activating the Nrf2 pathway (P<0.05); but Nrf2 inhibitor ML385 could reverse the above effects of GW501516 (P<0.05). GW501516 exerted similar effects to Nrf2 activator DMF in down-regulating the expression of C-caspase-3 and inhibiting the injury of PAECs under conditions of hypoxia (P<0.05). Moreover, Nrf2 inhibitor ML385 reversed the 163.com inhibition effects of GW501516 on PAECs injury (P<0.05). CONCLUSIONS GW501516 can relieve the hypoxia-induced injury of PAECs via the inhibition of oxidative stress, the mechanism of which may be associated with activating Nrf2.
ABSTRACT
OBJECTIVE: This study aimed to investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia, in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling. METHODS: PASMCs were incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) under the hypoxic condition. The proliferation was determined by a CCK-8 assay. The cell cycle progression was analyzed by flow cytometry. The expression of PPARδ, S phase kinase-associated protein 2 (Skp2), and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting. Then PASMCs were treated with 100 nmol/ L GW501516, 100 nmol/L mammalian target of rapamycin (mTOR) inhibitor rapamycin and/or 2 µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs. RESULTS: The presented data demonstrated that hypoxia reduced the expression of PPARδ in an oxygen concentration- and time-dependent manner, and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle. In accordance with these findings, GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs. Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation, arresting the cell cycle, regulating the expression of Skp2 and p27, and inactivating mTOR in hypoxia-exposed PASMCs. Moreover, MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs. CONCLUSION: GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.
ABSTRACT
The airway wall remodeling observed in asthma is associated with subepithelial fibrosis and enhanced activation of human bronchial fibroblasts (HBFs) in the fibroblast to myofibroblast transition (FMT), induced mainly by transforming growth factor-ß (TGF-ß). The relationships between asthma severity, obesity, and hyperlipidemia suggest the involvement of peroxisome proliferator-activated receptors (PPARs) in the remodeling of asthmatic bronchi. In this study, we investigated the effect of PPARδ ligands (GW501516 as an agonist, and GSK0660 as an antagonist) on the FMT potential of HBFs derived from asthmatic patients cultured in vitro. This report shows, for the first time, the inhibitory effect of a PPARδ agonist on the number of myofibroblasts and the expression of myofibroblast-related markers-α-smooth muscle actin, collagen 1, tenascin C, and connexin 43-in asthma-related TGF-ß-treated HBF populations. We suggest that actin cytoskeleton reorganization and Smad2 transcriptional activity altered by GW501516 lead to the attenuation of the FMT in HBF populations derived from asthmatics. In conclusion, our data demonstrate that a PPARδ agonist stimulates antifibrotic effects in an in vitro model of bronchial subepithelial fibrosis. This suggests its potential role in the development of a possible novel therapeutic approach for the treatment of subepithelial fibrosis during asthma.
Subject(s)
Asthma , PPAR delta , Humans , Transforming Growth Factor beta/metabolism , PPAR delta/metabolism , Transforming Growth Factor beta1/metabolism , Fibroblasts/metabolism , Asthma/metabolism , Bronchi/metabolism , Myofibroblasts/metabolism , Fibrosis , Cells, CulturedABSTRACT
Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of disease phenotypes which start with simple steatosis and lipid accumulation in the hepatocytes - a typical histological lesions characteristic. It may progress to non-alcoholic steatohepatitis (NASH) that is characterized by hepatic inflammation and/or fibrosis and subsequent onset of NAFLD-related cirrhosis and hepatocellular carcinoma (HCC). Due to the central role of the liver in metabolism, NAFLD is regarded as a result of and contribution to the metabolic abnormalities seen in the metabolic syndrome. Peroxisome proliferator-activated receptors (PPARs) has three subtypes, which govern the expression of genes responsible for energy metabolism, cellular development, inflammation, and differentiation. The agonists of PPARα, such as fenofibrate and clofibrate, have been used as lipid-lowering drugs in clinical practice. Thiazolidinediones (TZDs) - ligands of PPARγ, such as rosiglitazone and pioglitazone, are also used in the treatment of type 2 diabetes (T2D) with insulin resistance (IR). Increasing evidence suggests that PPARß/δ agonists have potential therapeutic effects in improving insulin sensitivity and lipid metabolism disorders. In addition, PPARs ligands have been considered as potential therapeutic drugs for hypertension, atherosclerosis (AS) or diabetic nephropathy. Their crucial biological roles dictate the significance of PPARs-targeting in medical research and drug discovery. Here, it reviews the biological activities, ligand selectivity and biological functions of the PPARs family, and discusses the relationship between PPARs and the pathogenesis of NAFLD and metabolic syndrome. This will open new possibilities for PPARs application in medicine, and provide a new idea for the treatment of fatty liver and related diseases.
Subject(s)
Carcinoma, Hepatocellular , Diabetes Mellitus, Type 2 , Liver Neoplasms , Metabolic Syndrome , Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/metabolism , Diabetes Mellitus, Type 2/metabolism , Metabolic Syndrome/metabolism , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Liver/metabolism , PPAR alpha/metabolism , Inflammation/metabolism , Lipids/therapeutic useABSTRACT
Lipid metabolism is essential in maintaining energy homeostasis in multicellular organisms. In vertebrates, the peroxisome proliferator-activated receptors (PPARs, NR1C) regulate the expression of many genes involved in these processes. Atlantic cod (Gadus morhua) is an important fish species in the North Atlantic ecosystem and in human nutrition, with a highly fatty liver. Here we study the involvement of Atlantic cod Ppar a and b subtypes in systemic regulation of lipid metabolism using two model agonists after in vivo exposure. WY-14,643, a specific PPARA ligand in mammals, activated cod Ppara1 and Ppara2 in vitro. In vivo, WY-14,643 caused a shift in lipid transport both at transcriptional and translational level in cod. However, WY-14,643 induced fewer genes in the fatty acid beta-oxidation pathway compared to that observed in rodents. Although GW501516 serves as a specific PPARB/D ligand in mammals, this compound activated cod Ppara1 and Ppara2 as well as Pparb in vitro. In vivo, it further induced transcription of Ppar target genes and caused changes in lipid composition of liver and plasma. The integrative approach provide a foundation for understanding how Ppars are engaged in regulating lipid metabolism in Atlantic cod physiology. We have shown that WY-14,643 and GW501516 activate Atlantic cod Ppara and Pparb, affect genes in lipid metabolism pathways, and induce changes in the lipid composition in plasma and liver microsomal membranes. Particularly, the combined transcriptomic, proteomics and lipidomics analyses revealed that effects of WY-14,643 on lipid metabolism are similar to what is known in mammalian studies, suggesting conservation of Ppara functions in mediating lipid metabolic processes in fish. The alterations in the lipid profiles observed after Ppar agonist exposure suggest that other chemicals with similar Ppar receptor affinities may cause disturbances in the lipid regulation of fish. Model organism: Atlantic cod (Gadus morhua). LSID: urn:lsid:zoobank.org:act:389BE401-2718-4CF2-BBAE-2E13A97A5E7B. COL Identifier: 6K72F.
ABSTRACT
BACKGROUND AND PURPOSE: Selective peroxisome proliferator-activated receptors (PPARs) are widely used to treat metabolic complications; however, the limited effect of PPARα agonists on glucose metabolism and the adverse effects associated with selective PPARγ activators have stimulated the development of novel pan-PPAR agonists to treat metabolic disorders. Here, we synthesized a new prenylated benzopyran (BP-2) and evaluated its PPAR-activating properties, anti-inflammatory effects and impact on metabolic derangements. EXPERIMENTAL APPROACH: BP-2 was used in transactivation assays to evaluate its agonism to PPARα, PPARß/δ and PPARγ. A parallel-plate flow chamber was employed to investigate its effect on TNFα-induced leukocyte-endothelium interactions. Flow cytometry and immunofluorescence were used to determine its effects on the expression of endothelial cell adhesion molecules (CAMs) and chemokines and p38-MAPK/NF-κB activation. PPARs/RXRα interactions were determined using a gene silencing approach. Analysis of its impact on metabolic abnormalities and inflammation was performed in ob/ob mice. KEY RESULTS: BP-2 displayed strong PPARα activity, with moderate and weak activity against PPARß/δ and PPARγ, respectively. In vitro, BP-2 reduced TNFα-induced endothelial ICAM-1, VCAM-1 and fractalkine/CX3CL1 expression, suppressed mononuclear cell arrest via PPARß/δ-RXRα interactions and decreased p38-MAPK/NF-κB activation. In vivo, BP-2 improved the circulating levels of glucose and triglycerides in ob/ob mice, suppressed T-lymphocyte/macrophage infiltration and proinflammatory markers in the liver and white adipose tissue, but increased the expression of the M2-like macrophage marker CD206. CONCLUSION AND IMPLICATIONS: BP-2 emerges as a novel pan-PPAR lead candidate to normalize glycemia/triglyceridemia and minimize inflammation in metabolic disorders, likely preventing the development of further cardiovascular complications.
Subject(s)
Metabolic Diseases , PPAR delta , PPAR-beta , Mice , Animals , PPAR gamma/metabolism , PPAR alpha/metabolism , PPAR-beta/metabolism , Tumor Necrosis Factor-alpha , Benzopyrans , NF-kappa B , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Inflammation/drug therapyABSTRACT
BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling-as most cardiac (genetic) diseases have a middle-age onset-and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor ß/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW). METHODS AND RESULTS: Monolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels. CONCLUSIONS: Collectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.
Subject(s)
Induced Pluripotent Stem Cells , Myocytes, Cardiac , Humans , Myocytes, Cardiac/metabolism , Induced Pluripotent Stem Cells/metabolism , Peroxisome Proliferator-Activated Receptors/metabolism , Cell Differentiation , Mitochondria/metabolismABSTRACT
Objective: To investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of primary rat proliferation of pulmonary artery smooth muscle cells ( PASMCs ) induced by hypoxia, in order to discover new drugs for the treatment and prevention of pulmonary vascular remodeling. Methods: The PASMCs in the control group were cultured with 21% oxygen, while the PASMCs in the hypoxia group were cultured with 3% oxygen to induce cell proliferation. PASMCs were incubated with GW501516 at the concentrations of 10, 30 and 100 nmol/L under hypoxic conditions for different time points (12, 24, and 48 h) to find out the appropriate concentrations of GW501516 for inhibition the proliferation. PASMCs were incubated with 100 nmol/L GW501516 and ( or ) protein kinase B (AKT) agonist SC79 for 24 h to explore related mechanisms of GW501516 in regulating the proliferation. The proliferation and DNA synthesis were determined by CCK-8 and BrdU kit. The cell cycle progression was analyzed by flow cytometry. The mRNA expressions of Cyclin D1 and the cyclin kinase inhibitor p27(p27) were measured by quantitative real-time PCR (RT-PCR). The expressions of PPARδ, total and phosphorylated forms AKT and glycogen synthase kinase 3ß (GSK3ß) were detected by Western blot. Results: Compared with the hypoxia group, PASMCs incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) for 12, 24, 48 h under hypoxic conditions could inhibit the proliferation and DNA synthesis, and the greatest level of suppression of proliferation was induced by GW501516 at the concentration of 100 nmol/L(Pï¼0.05 or Pï¼0.01). Compared with the control group, the expression of PPARδ was upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδ significantly(Pï¼0.01). Compared with the hypoxia group, 100 nmol/L GW501516 blocked the proliferation and DNA synthesis of PASMCs significantly(Pï¼0.01), increased the proportion of PASMCs in G0 /G1 phase while decreased the proportion of PASMCs in S phase and G2 /M phase(Pï¼0.05 or Pï¼0.01), markedly downregulated the mRNA expression of cyclin D1 and upregulated the mRNA expression of p27(Pï¼0.01), significantly inhibited the protein expressions of phosphorylated AKT and GSK3ß(Pï¼0.01). Compared with the 100 nmol/L GW501516 hypoxia group, AKT agonist SC79 reversed all the above effects of 100 nmol/L GW501516 on hypoxia stimulated PASMCs(Pï¼0.05 or Pï¼0.01). Conclusion: GW501516 inhibits hypoxia induced proliferation in PASMCs via inactivating AKT/GSK3ß signaling pathway.
Subject(s)
PPAR delta , Pulmonary Artery , Animals , Cell Hypoxia , Cell Proliferation , Cells, Cultured , Cyclin D1 , DNA , Glycogen Synthase Kinase 3 beta , Hypoxia , Myocytes, Smooth Muscle , Oxygen , Proto-Oncogene Proteins c-akt , RNA, Messenger , Rats , ThiazolesABSTRACT
According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.
Subject(s)
Doping in Sports/prevention & control , Substance Abuse Detection/methods , Thiazoles/analysis , Administration, Oral , Animals , Female , Horses , Male , Microsomes, Liver/metabolism , PPAR delta/agonists , Thiazoles/metabolism , Thiazoles/urineABSTRACT
Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARß (GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARß ligands possessed the strongest effect. The PPARß agonist, GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARß agonist GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for GW501516. The PPARß ligands, GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARß ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Astrocytes/metabolism , Interleukin-10/metabolism , Oxylipins/metabolism , PPAR gamma/agonists , PPAR-beta/agonists , Tumor Necrosis Factor-alpha/metabolism , Anilides/pharmacology , Animals , Astrocytes/drug effects , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Fenofibrate/pharmacology , Lipopolysaccharides/toxicity , MAP Kinase Kinase 4/metabolism , Oxazoles/pharmacology , PPAR gamma/antagonists & inhibitors , PPAR-beta/antagonists & inhibitors , Rats , Rats, Wistar , Rosiglitazone/pharmacology , Thiazoles/pharmacology , Tyrosine/analogs & derivatives , Tyrosine/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Gestational diabetes mellitus (GDM) is a disorder of glucose metabolism that occurs or is found for the first time during pregnancy. GDM is very harmful and urgently needs drug treatment to improve pregnancy outcome. PPARδ is involved in a variety of biological processes related to glycolipid metabolism in the body, suggesting that it may be closely related to insulin resistance and impaired glucose tolerance. The role of PPARδ agonist GW501516 in gestational diabetes has not been studied. METHODS: Firstly, the rat model of GDM was established. Then, fasting blood-glucose (FGB), fasting insulin (FINS), HOMA-islet resistance index (HOMA-IR) and insulin sensitivity index (ISI) of GDM rats treated with GW501516 were measured on day 3, day 10 and day 17. Glucose tolerance test was performed on the 20th day of gestation to measure glucose tolerance in rats. The expression of PPARδ and Angptl8 in islet tissues of rats was detected by Western blot and immunohistochemistry (IHC). Histopathological changes of islet were detected by HE stain; apoptosis rate of islet cells was detected by Tunel; and expression of apoptosis-related proteins in the cells was detected by Western blot. The biochemical kits were used to detect the expression of lipid metabolism-related factors in blood of GDM rats after the PPARδ agonist GW501516 treatment. Finally, the expression of SREBP-1c and GLUT2 in islet tissues was detected by RT-qPCR and IHC. RESULTS: The PPARδ agonist GW501516 decreased the expression of FGB, FINS and HOMA-IR in GDM rats, and we found that GW501516 decreased ISI in GDM rats. GW501516 increased glucose tolerance in GDM rats too. In GDM rats, the expression of PPARδ in islet decreased and the expression of Angptl8 increased, which was reversed by GW501516. In addition, we also found that GW501516 can improve the damaged islet tissue of GDM rats, reduce the apoptosis rate of islet cells and inhibit the expression of lipid metabolism-related factors in the blood. Finally, we found that GW501516 inhibited the expression of SREBP-1c and promoted the expression of GLUT2 in the islet tissue. CONCLUSION: The PPARδ agonist GW501516 could improve the blood glucose level, damaged islet tissue and increase the insulin content in the rats with GDM, possibly by regulating the SREBP-1c/GLUT2 pathway. Our study provided a new basis for clinical treatment of GDM in pregnant women with PPARδ agonist GW501516.
ABSTRACT
N-cadherin is a transmembrane glycoprotein expressed by mesenchymal origin cells and is located at the adherens junctions. It regulates also cell motility and contributes to cell signaling. In previous studies, we identified that its anomalous expression in bladder carcinoma was a tumor progression marker. A pharmacological approach to inhibit N-cadherin expression or to block its function could be relevant to prevent disease progression and metastasis development. The morphological exploration of T24 invasive bladder cancer cells by atomic force microscopy (AFM) revealed a spindle-like shape with fibrous structures. By engaging force spectroscopy with AFM tip functionalized with anti-E or anti-N-cadherin antibodies, results showed that T24 cells expressed only N-cadherin as also demonstrated by Western blotting and confocal microscopy. For the first time, we demonstrated by RTqPCR and Western blotting analyses that the peroxisome proliferator-activated receptor ß/δ (PPARß/δ) agonist GW501516 significantly decreased N-cadherin expression in T24 cells. Moreover, high non-cytotoxic doses of GW501516 inhibited confluent T24 cell wound healing closure. By using AFM, a more sensitive nanoanalytical method, we showed that the treatment modified the cellular morphology and diminished N-cadherin cell surface coverage through the decreasing of these adhesion molecule-mediated interaction forces. We observed a greater decrease of N-cadherin upon GW501516 exposure with AFM than that detected with molecular biology techniques. AFM was a complementary tool to biochemical techniques to perform measurements on living cells at the nanometer resolution level. Taken together, our data suggest that GW501516 could be an interesting therapeutic strategy to avoid bladder cancer cell spreading through N-cadherin decrease.
Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Epithelial-Mesenchymal Transition , Microscopy, Atomic Force/methods , PPAR delta/agonists , PPAR-beta/agonists , Thiazoles/pharmacology , Urinary Bladder Neoplasms/metabolism , Antigens, CD/ultrastructure , Cadherins/ultrastructure , Cell Line, Tumor , Cell Movement , Humans , Signal Transduction , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/ultrastructureABSTRACT
As a nuclear receptor, ligand binding and activated PPARδ (peroxisome-proliferator-activated receptor δ) plays an important role in regulation of inflammation, metabolism and cancer, while it is unclear the effect of metformin on PPARδ-mediated cancer cell metabolism. Here we found that PPARδ agonist GW501516 significantly increased Glut1 (Glucose transporter 1) and SLC1A5 (solutecarrier family 1 member 5) gene and protein expressions in HCT-116, SW480, HeLa, and MCF-7 cancer cell lines, while metformin inhibited this event, which was associated with metformin-mediated inhibition of PPARδ activity in response to GW501516. Importantly, GW501516 inhibited the binding of PPARδ to AMPK, while metformin reversed this process. Metformin inhibited Glut1 and SLC1A5 expressions leading to reduced influx of glucose and glutamine in cancer cells, which is associated with reduced tumor growth. These findings suggest that metformin inhibited PPARδ agonist GW501516-induced cancer cell metabolism and tumor growth.
Subject(s)
Amino Acid Transport System ASC/genetics , Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Glucose Transporter Type 1/genetics , Metformin/pharmacology , Minor Histocompatibility Antigens/genetics , PPAR delta/agonists , Thiazoles/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Glucose/metabolism , Glutamine/metabolism , Humans , Mice , Thiazoles/pharmacology , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Previous reports have shown that PPARß/δ agonists ameliorate insulin resistance associated with type 2 diabetes mellitus (T2DM). To determine the role of PPARß/δ in tumor necrosis factor α (TNFα)-mediated insulin resistance, we investigated expression levels of adiponectin and insulin receptor (IR) in response to treatment with the PPARß/δ agonist GW501516 with or without TNFα, a proinflammatory cytokine, in differentiated 3T3-L1 adipocytes. GW501516 induced adipocyte differentiation and the expression of adiponectin in a dose-dependent manner in differentiated adipocytes. TNFα treatment reduced adiponectin expression at the end of differentiation. This effect was reversed by GW501516 co-treatment with TNFα. TNFα treatment decreased adipogenic marker genes such as PPARγ, aP2, resistin, and GLUT4, and GW501516 reversed the effects of TNFα. GW501516 treatment increased the expression of insulin receptor and inhibited TNFα-mediated repression of insulin receptor. Our results showed that GW501516 abrogated TNFα-induced insulin resistance. In summary, our study demonstrated that the PPARß/δ agonist, GW501516 reversed TNFα-induced decreases in adipocyte differentiation and adiponectin expression, and improved insulin sensitivity by increasing the expression of insulin receptor. Therefore, PPARδ may be a promising therapeutic target for treatment of insulin resistance in patients with T2DM.
Subject(s)
Adiponectin/metabolism , PPAR delta/agonists , PPAR-beta/agonists , Receptor, Insulin/metabolism , Thiazoles/pharmacology , Tumor Necrosis Factor-alpha/metabolism , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Insulin Resistance , Mice , PPAR delta/metabolism , PPAR-beta/metabolismABSTRACT
As a nuclear receptor, peroxisome proliferator-activated receptor-δ (PPARδ) plays a critical role in regulating inflammation and cancer, while it is still unclear the mechanism of PPARδ agonist GW501516 on colitis-associated colorectal cancer. Here we found that GW501516 significantly enhanced colitis-associated colorectal cancer in AOM/DSS-induced mice. In addition, PPARδ agonist GW501516 enhanced pro-inflammatory gene expressions (COX-2, IL-6, IL-8 and MCP-1) in inflamed colon. Further analysis showed that GW501516 increased the expressions of Glut1 and SLC1A5 in colon cancer cells as well as AOM/DSS-induced colorectal tumors. These findings revealed a new mechanism of PPARδ agonist GW501516-mediated colitis-associated colorectal cancer.
Subject(s)
Colitis/complications , Colorectal Neoplasms/complications , Colorectal Neoplasms/pathology , PPAR delta/agonists , Thiazoles/pharmacology , Animals , Carcinogenesis/chemically induced , Cell Line, Tumor , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) ß/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.
ABSTRACT
Activation of peroxisome proliferator-activated receptor ß/δ (PPARß/δ) had been linked to inhibition on the proliferation and apoptosis in a few cancer cell lines. However, limited data exists regarding the role of PPARß/δ in nasopharyngeal carcinoma (NPC). This study was undertaken to determine the effect of PPARß/δ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the human NPC cell lines. Gene and protein expression of PPARß/δ were reduced specifically in the poor- and un-differentiated NPC cell lines as compared with the control NP-69 cells. Ligand activation of PPARß/δ by GW501516, a specific PPARß/δ selective agonist, inhibited cell proliferation and colony formation strikingly, and induced a G2/M phase arrest in the EBV positive undifferentiated NPC C666-1 cells relative to the control cells. Moreover, GW501516 induced C666-1 cell apoptosis in a caspase and BAX dependent manner. In accordance with the in vitro result, GW501516 significantly suppressed the ectopic NPC xenograft tumorigenicity that derived from the C666-1 NPC cells in BALB/c nu/nu mice. This effect is greatly associated with its inhibition on the gene and protein expression of integrin-linked kinase (ILK) through activation of the AMPKα-dependent signaling pathways. Collectively, we showed that PPARß/δ expression is in reverse correlation with the degree of differentiation in the NPC cell lines, and revealed the anti-tumorigenic effects of GW501516 in NPC cells by activation of AMPKα. This study suggested that PPARß/δ targeting molecules may be useful for the poor-, and particularly un-differentiated NPC chemoprevention.
ABSTRACT
Exercise exerts significant effects on the prevention and treatment of many diseases. However, even though some of the key regulators of training adaptation in skeletal muscle have been identified, this biological program is still poorly understood. Accordingly, exercise-based pharmacological interventions for many muscle wasting diseases and also for pathologies that are triggered by a sedentary lifestyle remain scarce. The most efficacious compounds that induce muscle hypertrophy or endurance are hampered by severe side effects and are classified as doping. In contrast, dietary supplements with a higher safety margin exert milder outcomes. In recent years, the design of pharmacological agents that activate the training program, so-called "exercise mimetics", has been proposed, although the feasibility of such an approach is highly debated. In this review, the most recent insights into key regulatory factors and therapeutic approaches aimed at leveraging exercise adaptations are discussed.